Single cell analysis core

项目总结 阿尔茨海默病(AD)是一种破坏性的神经退行性疾病，其最大的单一风险 因素是年龄。目前，这种疾病还没有有效的治疗方法。因为AD要求这样一个 给患者及其家人带来巨大的经济和情感损失，迫切需要开发新的 了解和管理AD的方法。核心D在本计划项目的背景下具有重要意义 因为它提供了一种新的方法来理解衰老是如何通过小的积累 大量的衰老细胞，可能导致与AD相关的神经元变性。 单细胞图谱使我们能够表征罕见的细胞亚群，如衰老细胞， 可能逃脱大量种群常规分析的检测。几十年来，我们推断 通过使用均质的组织、细胞或甚至整个组织来分析基因表达 有机体。这种方法导致了许多发现，尽管随着我们开始的时候回报越来越少 了解少量细胞群对周围细胞、整个组织和/或 全身环境。为了实现这一PPG中计划的项目，核心D将制定四个具体目标。 在目标1中，我们将描述星形胶质细胞的细胞大小和衰老基因表达谱， 小胶质细胞和神经元在培养中诱导衰老，并测定染色质的变化 可通过ATAC-seq访问。我们假设诱导细胞衰老会导致异质性 细胞大小的阵列以及不同大小的衰老细胞具有不同的基因表达谱。目标2将 确定从衰老和非衰老的星形胶质细胞、神经元、 小胶质细胞反映了诱导衰老的完整单细胞的基因表达模式。基于一个 一些使用其他非衰老细胞类型的研究，我们假设从这些细胞中分离出的细胞核将， 在很大程度上反映了完整单细胞中的基因表达模式。目标3将开发和应用一种新的 用于体内验证单细胞衰老信号的技术--空间转录学 目标1和2。我们将开发这项技术来研究神经元和衰老星形胶质细胞之间的相互作用。 在目标4中，我们将把目标1-3中开发的方法和工作流程应用于每个范例 在项目1-3中测试。已建立特定的蛋白质(核心C)和基因表达签名 在AIMS 1和AIMS 2中，由于衰老导致的不同细胞类型，这个最终目标将使用一种新的验证方法来 在组织切片或细胞模型中确认我们独特的单细胞表达特征 项目。 总体而言，我们的核心将提供强大的新方法，以更好地理解衰老在 在单个细胞水平上的多个AD模型系统。

PROJECT SUMMARY Alzheimer’s disease (AD) is a devastating neurodegenerative disease for which the largest single risk factor is age. At present, there are no effective treatments for this disease. Because AD exacts such an enormous economic and emotional toll on patients and their families, there is an urgent need to develop new approaches to understand and manage AD. Core D is significant in the context of this program project because it provides a new approach to understanding how aging, through the accumulation of a small population of senescent cells, might drive the neuronal degeneration associated with AD. Single-cell profiling allows us to characterize rare subpopulations of cells, such as senescent cells, that might escape detection by conventional analyses of bulk populations. For decades, we inferred the presence and properties of senescent cells by profiling gene expression using homogenized tissues, cells or even entire organisms. This approach led to many discoveries, although increasingly with diminishing returns as we began to appreciate the role that small populations of cells can have on surrounding cells, entire tissues and/or the systemic milieu. To enable the planned projects in this PPG, Core D will develop four specific aims. In AIM 1, we will characterize cell size and senescent gene expression profiles of astrocytes, microglia, and neurons induced to senescence in culture, and determine changes in chromatin accessibility via ATAC-seq. We hypothesize that induced cellular senescence results in a heterogeneous array of cell sizes and that senescent cells of different sizes have different gene expression profiles. AIM 2 will determine the extent to which nuclei isolated from senescent and non-senescent astrocytes, neurons, and microglia reflect the gene expression pattern of intact single cells induced to senesce. Based on a handful of studies using other non-senescent cell types, we hypothesize that nuclei isolated from these cells will, to a large extent, reflect the gene expression pattern in intact single cells. AIM 3 will develop and apply a new technology – spatial transcriptomics -- for in vivo validation of single-cell senescent signatures from Aims 1 and 2. We will develop this technology to study the interplay between neurons and senescent astrocytes. In AIM 4, we will apply methods and workflows developed in Aims 1–3 to each of the paradigms being tested in Projects 1–3. Having established specific protein (Core C) and gene expression signatures for different cell types as a result of senescence in aims 1 and 2, this final aim will use a novel validation method to confirm our unique single cell expression signatures in tissue sections or cell models where applicable in each project. Overall our Core will provide powerful new approaches to better understand the role of senescence in multiple model systems of AD at the level of the single cell.